Correction: The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

[This corrects the article DOI: 10.1371/journal.pgen.1010766.].

The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM.

GIGANTEA Is a Negative Regulator of Abscisic Acid Transcriptional Responses and Sensitivity in Arabidopsis.

Transcriptional reprogramming plays a key role in drought stress responses, preceding the onset of morphological and physiological acclimation. The best-characterized signal regulating gene expression in response to drought is the phytohormone abscisic acid (ABA). ABA-regulated gene expression, biosynthesis and signaling are highly organized in a diurnal cycle, so that ABA-regulated physiological traits occur at the appropriate time of day. The mechanisms that underpin such diel oscillations in ABA signals are poorly characterized. Here we uncover GIGANTEA (GI) as a key gatekeeper of ABA-regulated transcriptional and physiological responses. Time-resolved gene expression profiling by RNA sequencing under different irrigation scenarios indicates that gi mutants produce an exaggerated ABA response, despite accumulating wild-type levels of ABA. Comparisons with ABA-deficient mutants confirm the role of GI in controlling ABA-regulated genes, and the analysis of leaf temperature, a read-out for transpiration, supports a role for GI in the control of ABA-regulated physiological processes. Promoter regions of GI/ABA-regulated transcripts are directly targeted by different classes of transcription factors (TFs), especially PHYTOCHROME-INTERACTING FACTOR and -BINDING FACTOR, together with GI itself. We propose a model whereby diel changes in GI control oscillations in ABA responses. Peak GI accumulation at midday contributes to establishing a phase of reduced ABA sensitivity and related physiological responses, by gating DNA binding or function of different classes of TFs that cooperate or compete with GI at target regions.

The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells.

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

Time-Course Transcriptome Analysis of Arabidopsis Siliques Discloses Genes Essential for Fruit Development and Maturation.

Fruits protect the developing seeds of angiosperms and actively contribute to seed dispersion. Furthermore, fruit and seed development are highly synchronized and require exchange of information between the mother plant and the developing generations. To explore the mechanisms controlling fruit formation and maturation, we performed a transcriptomic analysis on the valve tissue of the Arabidopsis (Arabidopsis thaliana) silique using RNA sequencing. In doing so, we have generated a data set of differentially regulated genes that will help to elucidate the molecular mechanisms that underpin the initial phase of fruit growth and, subsequently, trigger fruit maturation. The robustness of our data set has been tested by functional genomic studies. Using a reverse genetics approach, we selected 10 differentially expressed genes and explored the consequences of their disruption for both silique growth and senescence. We found that genes contained in our data set play essential roles in different stages of silique development and maturation, indicating that our transcriptome-based gene list is a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation in Arabidopsis.

Abscisic Acid Is a Major Regulator of Grape Berry Ripening Onset: New Insights into ABA Signaling Network.

Grapevine is a world-wide cultivated economically relevant crop. The process of berry ripening is non-climacteric and does not rely on the sole ethylene signal. Abscisic acid (ABA) is recognized as an important hormone of ripening inception and color development in ripening berries. In order to elucidate the effect of this signal at the molecular level, pre-véraison berries were treated ex vivo for 20 h with 0.2 mM ABA and berry skin transcriptional modulation was studied by RNA-seq after the treatment and 24 h later, in the absence of exogenous ABA. This study highlighted that a small amount of ABA triggered its own biosynthesis and had a transcriptome-wide effect (1893 modulated genes) characterized by the amplification of the transcriptional response over time. By comparing this dataset with the many studies on ripening collected within the grapevine transcriptomic compendium Vespucci, an extended overlap between ABA- and ripening modulated gene sets was observed (71% of the genes), underpinning the role of this hormone in the regulation of berry ripening. The signaling network of ABA, encompassing ABA metabolism, transport and signaling cascade, has been analyzed in detail and expanded based on knowledge from other species in order to provide an integrated molecular description of this pathway at berry ripening onset. Expression data analysis was combined with in silico promoter analysis to identify candidate target genes of ABA responsive element binding protein 2 (VvABF2), a key upstream transcription factor of the ABA signaling cascade which is up-regulated at véraison and also by ABA treatments. Two transcription factors, VvMYB143 and VvNAC17, and two genes involved in protein degradation, Armadillo-like and Xerico-like genes, were selected for in vivo validation by VvABF2-mediated promoter trans-activation in tobacco. VvNAC17 and Armadillo-like promoters were induced by ABA via VvABF2, while VvMYB143 responded to ABA in a VvABF2-independent manner. This knowledge of the ABA cascade in berry skin contributes not only to the understanding of berry ripening regulation but might be useful to other areas of viticultural interest, such as bud dormancy regulation and drought stress tolerance.

SUMO proteases OTS1 and 2 control filament elongation through a DELLA-dependent mechanism.

KEY MESSAGE: SUMOylation and anther growth. During fertilization, stamen elongation needs to be synchronized with pistil growth. The phytohormone gibberellic acid (GA) promotes stamen growth by stimulating the degradation of growth repressing DELLA proteins. DELLA accumulation is negatively regulated by GAs through the ubiquitin-proteasome system. In Arabidopsis thaliana, a proportion of DELLAs is also conjugated to the small ubiquitin-like modifier (SUMO) protein, which stabilizes DELLAs. Increased DELLA levels occur in the SUMO protease-deficient OVERLY TOLERANT TO SALT 1 and 2 (ots1 ots2) double mutants, especially under salt stress conditions. Here, we show that OTS genes play a redundant role in the control of plant fertility under non-stress conditions. Mutants of ots1 ots2 display reduced fertility compared with the wild type, owing to reduced stamen elongation. Stamen growth, pollination rate and seed production are restored in ots1 ots2 della mutants, thus linking OTS1 function to the control of DELLA activity in the context of filament elongation. OTS levels appear to be developmentally regulated as OTS1/2 transcript upregulation during stamen development overlaps with GAs accumulations. We propose that OTS genes enable synchronization of stamen development by facilitating DELLA degradation at a specific developmental stage.

ABA-dependent control of GIGANTEA signalling enables drought escape via up-regulation of FLOWERING LOCUS T in Arabidopsis thaliana.

One strategy deployed by plants to endure water scarcity is to accelerate the transition to flowering adaptively via the drought escape (DE) response. In Arabidopsis thaliana, activation of the DE response requires the photoperiodic response gene GIGANTEA (GI) and the florigen genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF). The phytohormone abscisic acid (ABA) is also required for the DE response, by promoting the transcriptional up-regulation of the florigen genes. The mode of interaction between ABA and the photoperiodic genes remains obscure. In this work we use a genetic approach to demonstrate that ABA modulates GI signalling and consequently its ability to activate the florigen genes. We also reveal that the ABA-dependent activation of FT, but not TSF, requires CONSTANS (CO) and that impairing ABA signalling dramatically reduces the expression of florigen genes with little effect on the CO transcript profile. ABA signalling thus has an impact on the core genes of photoperiodic signalling GI and CO by modulating their downstream function and/or activities rather than their transcript accumulation. In addition, we show that as well as promoting flowering, ABA simultaneously represses flowering, independent of the florigen genes. Genetic analysis indicates that the target of the repressive function of ABA is the flowering-promoting gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a transcription factor integrating floral cues in the shoot meristem. Our study suggests that variations in ABA signalling provide different developmental information that allows plants to co-ordinate the onset of the reproductive phase according to the available water resources.

A novel role for STOMATAL CARPENTER 1 in stomata patterning.

BACKGROUND: Guard cells (GCs) are specialised cells within the plant epidermis which form stomatal pores, through which gas exchange can occur. The GCs derive through a specialised lineage of cell divisions which is specified by the transcription factor SPEECHLESS (SPCH), the expression of which can be detected in undifferentiated epidermal cells prior to asymmetric division. Other transcription factors may act before GC specification and be required for correct GC patterning. Previously, the DOF transcription factor STOMATAL CARPENTER 1 (SCAP1) was shown to be involved in GC function, by activating a set of GC-specific genes required for GC maturation and activity. It is thus far unknown whether SCAP1 can also affect stomatal development. RESULTS: Here we show that SCAP1 expression can also be observed in young leaf primordia, before any GC differentiation occurs. The study of transgenic plants carrying a proSCAP1:GUS-GFP transcriptional fusion, coupled with qPCR analyses, indicate that SCAP1 expression peaks in a temporal window which is coincident with expression of stomatal patterning genes. Independent scap1 loss-of-function mutants have a reduced number of GCs whilst SCAP1 over expression lines have an increased number of GCs, in addition to altered GC distribution and spacing patterns. The study of early markers for stomatal cell lineage in a background carrying gain-of-function alleles of SCAP1 revealed that, compared to the wild type, an increased number of protodermal cells are recruited in the GC lineage, which is reflected in an increased number of meristemoids. CONCLUSIONS: Our results suggest an early role for SCAP1 in GC differentiation. We propose that a function of SCAP1 is to integrate different aspects of GC biology including specification, spacing, maturation and function.

Genome-Wide Transcriptome Analysis During Anthesis Reveals New Insights into the Molecular Basis of Heat Stress Responses in Tolerant and Sensitive Rice Varieties.

Rice is one of the main food crops in the world. In the near future, yield is expected to be under pressure due to unfavorable climatic conditions, such as increasing temperatures. Therefore, improving rice germplasm in order to guarantee rice production under harsh environmental conditions is of top priority. Although many physiological studies have contributed to understanding heat responses during anthesis, the most heat-sensitive stage, molecular data are still largely lacking. In this study, an RNA-sequencing approach of heat- and control-treated reproductive tissues during anthesis was carried out using N22, one of the most heat-tolerant rice cultivars known to date. This analysis revealed that expression of genes encoding a number of transcription factor families, together with signal transduction and metabolic pathway genes, is repressed. On the other hand, expression of genes encoding heat shock factors and heat shock proteins was highly activated. Many of these genes are predominantly expressed at late stages of anther development. Further physiological experiments using heat-tolerant N22 and two sensitive cultivars suggest that reduced yield in heat-sensitive plants may be associated with poor pollen development or production in anthers prior to anthesis. In parallel, induction levels of a set of heat-responsive genes in these tissues correlated well with heat tolerance. Altogether, these findings suggest that proper expression of protective chaperones in anthers is needed before anthesis to overcome stress damage and to ensure fertilization. Genes putatively controlling this process were identified and are valuable candidates to consider for molecular breeding of highly productive heat-tolerant cultivars.

Comparative analysis of fruit aroma patterns in the domesticated wild strawberries "Profumata di Tortona" (F. moschata) and "Regina delle Valli" (F. vesca).

Strawberry is one of the most valued fruit worldwide. Modern cultivated varieties (Fragaria × ananassa) exhibit large fruits, with intense color and prolonged shell life. Yet, these valuable traits were attained at the cost of the intensity and the variety of the aroma of the berry, two characteristics highly appreciated by consumers. Wild species display smaller fruits and reduced yield compared with cultivated varieties but they accumulate broader and augmented blends of volatile compounds. Because of the large diversity and strength of aromas occurring in natural and domesticated populations, plant breeders regard wild strawberries as important donors of novel scented molecules. Here we report a comprehensive metabolic map of the aroma of the wild strawberry Profumata di Tortona (PdT), an ancient clone of F. moschata, considered as one of the most fragrant strawberry types of all. Comparison with the more renowned woodland strawberry Regina delle Valli (RdV), an aromatic cultivar of F. vesca, revealed a significant enrichment in the total level of esters, alcohols and furanones and a reduction in the content of ketones in in the aroma of PdT berries. Among esters, particularly relevant was the enhanced accumulation of methyl anthranilate, responsible for the intensive sweetish impression of wild strawberries. Interestingly, increased ester accumulation in PdT fruits correlated with enhanced expression of the Strawberry Alcohol Acyltransferase (SAAT) gene, a key regulator of flavor biogenesis in ripening berries. We also detected a remarkable 900-fold increase in the level of mesifurane, the furanone conferring the typical caramel notes to most wild species.

Small Ubiquitin-like Modifier protein SUMO enables plants to control growth independently of the phytohormone gibberellin.

Plants survive adverse conditions by modulating their growth in response to a changing environment. Gibberellins (GAs) play a key role in these adaptive responses by stimulating the degradation of growth-repressing DELLA proteins. GA binding to its receptor GID1 enables association of GID1 with DELLAs. This leads to the ubiquitin-mediated proteasomal degradation of DELLAs and consequently growth promotion. We report that DELLA-dependent growth control can be regulated independently of GA. We demonstrate that when a proportion of DELLAs is conjugated to the Small Ubiquitin-like Modifier (SUMO) protein, the extent of conjugation increases during stress. We identify a SUMO-interacting motif in GID1 and demonstrate that SUMO-conjugated DELLA binds to this motif in a GA-independent manner. The consequent sequestration of GID1 by SUMO-conjugated DELLAs leads to an accumulation of non-SUMOylated DELLAs, resulting in beneficial growth restraint during stress. We conclude that plants have developed a GA-independent mechanism to control growth.

Environmental stress and flowering time: the photoperiodic connection.

Plants maximize their chances to survive adversities by reprogramming their development according to environmental conditions. Adaptive variations in the timing to flowering reflect the need for plants to set seeds under the most favorable conditions. A complex network of genetic pathways allows plants to detect and integrate external (e.g., photoperiod and temperature) and/or internal (e.g., age) information to initiate the floral transition. Furthermore different types of environmental stresses play an important role in the floral transition. The emerging picture is that stress conditions often affect flowering through modulation of the photoperiodic pathway. In this review we will discuss different modes of cross talk between stress signaling and photoperiodic flowering, highlighting the central role of the florigen genes in this process.

The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells.

Plants have evolved different strategies to resist drought, of which the best understood is the abscisic acid (ABA)-induced closure of stomatal pores to reduce water loss by transpiration. The availability of useful promoters that allow for precise spatial and temporal control of gene expression in stomata is essential both for investigating stomatal regulation in model systems and for biotechnological applications in field crops. Previous work indicated that the regulatory region of the transcription factor AtMYB60 specifically drives gene expression in guard cells of Arabidopsis, although its activity is rapidly down-regulated by ABA. Here, the activity of the full-length and minimal AtMYB60 promoters is reported in rice (Oryza sativa), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum), using a reporter gene approach. In rice, the activity of both promoters was completely abolished, whereas it was spatially restricted to guard cells in tobacco and tomato. To overcome the negative effect of ABA on the AtMYB60 promoter, a chimeric inducible system was developed, which combined the cellular specificity of the AtMYB60 minimal promoter with the positive responsiveness to dehydration and ABA of the rd29A promoter. Remarkably, the synthetic module specifically up-regulated gene expression in guard cells of Arabidopsis, tobacco, and tomato in response to dehydration or ABA. The comparative analysis of different native and synthetic regulatory modules derived from the AtMYB60 promoter offers new insights into the functional conservation of the cis-mechanisms that mediate gene expression in guard cells in distantly related dicotyledonous species and provides novel tools for modulating stomatal activity in plants.

GIGANTEA enables drought escape response via abscisic acid-dependent activation of the florigens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS.

Modulation of the transition to flowering plays an important role in the adaptation to drought. The drought-escape (DE) response allows plants to adaptively shorten their life cycle to make seeds before severe stress leads to death. However, the molecular basis of the DE response is unknown. A screen of different Arabidopsis (Arabidopsis thaliana) flowering time mutants under DE-triggering conditions revealed the central role of the flower-promoting gene GIGANTEA (GI) and the florigen genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) in the DE response. Further screens showed that the phytohormone abscisic acid is required for the DE response, positively regulating flowering under long-day conditions. Drought stress promotes the transcriptional up-regulation of the florigens in an abscisic acid- and photoperiod-dependent manner, so that early flowering only occurs under long days. Along with the florigens, the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 is also up-regulated in a similar fashion and contributes to the activation of TSF. The DE response was recovered under short days in the absence of the floral repressor SHORT VEGETATIVE PHASE or in GI-overexpressing plants. Our data reveal a key role for GI in connecting photoperiodic cues and environmental stress independently from the central FT/TSF activator CONSTANS. This mechanism explains how environmental cues may act upon the florigen genes in a photoperiodically controlled manner, thus enabling plastic flowering responses.

Challenges and perspectives to improve crop drought and salinity tolerance.

Drought and high salinity are two major abiotic stresses affecting crop productivity. Therefore, the development of crops better adapted to cope with these stresses represents a key goal to ensure global food security to an increasing world population. Although many genes involved in the response to these abiotic stresses have been extensively characterised and some stress tolerant plants developed, the success rate in producing stress-tolerant crops for field conditions has been thus far limited. In this review we discuss different factors hampering the successful transfer of beneficial genes from model species to crops, emphasizing some limitations in the phenotypic characterisation and definition of the stress tolerant plants developed so far. We also highlight some technological advances and different approaches that may help in developing cultivated stress tolerant plants.

DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter.

BACKGROUND: We previously demonstrated that the Arabidopsis thaliana AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. AtMYB60 is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA. RESULTS: To investigate the molecular mechanisms governing AtMYB60 expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of AtMYB60 promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the AtMYB60 promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of AtMYB60 expression. CONCLUSIONS: These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.

The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress.

BACKGROUND: Under drought, plants accumulate the signaling hormone abscisic acid (ABA), which induces the rapid closure of stomatal pores to prevent water loss. This event is trigged by a series of signals produced inside guard cells which finally reduce their turgor. Many of these events are tightly regulated at the transcriptional level, including the control exerted by MYB proteins. In a previous study, while identifying the grapevine R2R3 MYB family, two closely related genes, VvMYB30 and VvMYB60 were found with high similarity to AtMYB60, an Arabidopsis guard cell-related drought responsive gene. RESULTS: Promoter-GUS transcriptional fusion assays showed that expression of VvMYB60 was restricted to stomatal guard cells and was attenuated in response to ABA. Unlike VvMYB30, VvMYB60 was able to complement the loss-of-function atmyb60-1 mutant, indicating that VvMYB60 is the only true ortholog of AtMYB60 in the grape genome. In addition, VvMYB60 was differentially regulated during development of grape organs and in response to ABA and drought-related stress conditions. CONCLUSIONS: These results show that VvMYB60 modulates physiological responses in guard cells, leading to the possibility of engineering stomatal conductance in grapevine, reducing water loss and helping this species to tolerate drought under extreme climatic conditions.